Salinity reduces carbon assimilation and the harvest index of cassava plants ( Manihot esculenta Crantz )

This study was developed to evaluate the effects of salinity on the growth and gas exchange of cassava plants, cultivar Verdinha. The four concentrations of NaCl (mM) were as follows: 0, 20, 40, and 60. Under salinity, the lowest concentration of Na ions was observed in the tuberous roots; however, the dry matter of tuberous roots was reduced with an application of just 20 mM NaCl. The harvest index was reduced 50% with the highest salt concentration. Salinity reduced carbon assimilation (A), stomatal conductance (gs), transpiration, and the instantaneous water use efficiency. The correlation between gs and A was high and positive, showing that stomatal movement was one of the responsible for the lower A. Under salt stress, there was an increase in intercellular CO2 concentration, indicating the impairment of carbon metabolism. Based on the reduction of dry matter of the tuberous roots (reduction of 81% under 60 mM NaCl), it was concluded that cassava is sensitive to salinity. The growth of shoots and the absorbing roots were minimally affected by salinity, even in the situation where A was reduced; therefore, the sensitivity of cassava was related to the high sensitivity of the tuberous roots to the ionic and/or osmotic effects of salinity. Thus, tuberous roots can be the target organ in studies that aim to improve the tolerance of cassava to salinity.


Introduction
The inadequate management of irrigation water, coupled with the intensive use of soluble fertilizers with a high salt index, has contributed to the increase of agricultural areas with salinity problems.Indeed, in addition to natural salinization, a significant proportion of areas incorporated into the production process have become salty due to: (i) the replacement of evergreen vegetation with annual crops, which causes top soil erosion; and (ii) inadequate irrigation, which increases salt concentrations in the root zone (Munns & Tester, 2008).This is particularly important in arid and semi-arid regions due to the lack of rainfall and high evaporative demand, which hinders the leaching of salts found in the soil arable layer.The salinity problem is increasing considerably, and there are estimates that worldwide more than one billion hectares are affected by salinity (Wicke et al., 2011).Some authors consider salinity the main environmental factor limiting plant growth and productivity worldwide (Dasgupta, Nandy, & Das, 2013;Gupta & Huang, 2014).Thus, by studying the negative effects of salinity on the physiology and growth of crops, it is possible to generate information that helps improve crop yields when cultivated in this adverse condition.Crop species show a spectrum of responses to NaCl, though the majority has their growth reduced by salinity (Manchanda & Garg, 2008).Salt stress involves changes in various physiological and metabolic processes depending on the species or genotype, salt and nutrient composition in the solution, environmental conditions (Tedeschi et al., 2017), and stress severity and duration (Gupta & Huang, 2014).According Munns and Tester (2008), soil salinity inhibits plant growth for two reasons: (i) a reduction in a plant's ability to absorb water, which is the osmotic effect or water-deficit induced by salinity; and because (ii) NaCl ions can enter the transpiration stream and eventually harm a plant's cell metabolism.In fact, the growth reduction induced by NaCl has also been correlated with low photosynthate supply to the sink organs as a consequence of the reduction in the photosynthetic capacity under these conditions (Eisa, Hussin, Geissler, & Koyro, 2012).Decreases in photosynthesis can occur by two main mechanisms: (i) restricted diffusion (CO 2 flux to carboxylation sites) caused by decreases in stomatal and internal conductance; and (ii) the inhibition of the metabolic potential for photosynthesis (Pérez-López, Robredo, Lacuesta, Mena-Petite, & Muñoz-Rueda, 2012).Plants develop several physiological and biochemical mechanisms in order to survive in soil with high salt concentrations.In a comprehensive review, Manchanda and Garg (2008) indicated that the main mechanisms include ion absorption, transport, homeostasis and compartmentalization, the biosynthesis of compatible solutes and osmoprotectants, the synthesis of antioxidant compounds, the activation of antioxidant enzyme, polyamine synthesis, the generation of nitric oxide (NO) and hormonal modulation.For different species, it is important to know these mechanisms in order to develop technologies that reduce the negative effects of salinity.
Cassava is considered a robust species with tolerance to acidic and low fertility soils.It also shows a high level of tolerance to high temperatures and drought.Good production capacity, even in unfavorable conditions, has led researchers to suggest cassava as a strategic crop option to mitigate hunger in many poor regions of the world (Burns, Gleadow, Cliff, Zacarias, & Cavagnaro, 2010).However, few studies have been developed to evaluate the response of cassava to salinity.Anon (1976), cited by Hawker and Smith (1982), described a field study in which cassava had an approximately 50% decrease in yield at a salinity level as low as 6 to 8 mM NaCl (0.7 mS cm -1 ).Carretero, Cantos, García, and Troncoso (2007) observed that salinity adversely affected survival, development, and mineral composition of three cassava clones.In research conducted by Hawker and Smith (1982), salinity seriously affected cassava growth; because of this, they considered it a moderately salt sensitive species.Recently, studies have been exploring salinity effects with a focus on the proteome and genome analysis of cassava (Costa et al., 2011;An et al., 2014;Santa Brígida et al., 2014).
Although the salinity problem is growing, the literature regarding cassava's response to salinity is scarce (Setter andFregene, 2007, Santa Brígida et al., 2014).However, cassava has shown to have potential to be improved for salt stress through screening and selection (Carretero et al., 2007).The objective of this study was to evaluate the effects of NaCl concentrations on cassava growth, leaf gas exchange, and dry matter accumulation.

Experimental conditions and plant culture
The study was carried out in a greenhouse at Embrapa Mandioca e Fruticultura located in Cruz das Almas, Bahia State, Brazil; the greenhouse was equipped with the automatic control of temperature (30°C ± 2°C) and relative humidity (64% ± 2%).The cultivar Verdinha (Manihot esculenta Crantz, BGM 116) was chosen for the experiment.This variety has good yield potential, which was determined by the National Center for Research on Cassava and Tropical Fruits (CNPMF/EMBRAPA), for commercial plantations in some areas of the Brazilian Northeast region.Two stem cuttings (15 cm long) were planted in pots with a 14 L capacity.Before planting, the pots were filled with a mixture of sand, perlite, and vegetable substrate (composed of 60% pinus bark + 30% granulated coconut fiber + 10% vermiculite) in equal proportions.During the first 15 days, the pots were watered twice a day with tap water until field capacity.After this period, the less vigorous plant was discarded, and the experiment proceeded with only one plant per pot.After thinning, the substrate was fertilized with a growth nutrient solution, modified from the solution used by Cruz, Alves, LeCain, Ellis, and Morgan (2014)     A considerable part of absorbed Na + was retained in the absorption roots; however, large amounts of Na + were translocated to the shoot.The stem + petiole retained most of the translocated Na + , preventing it from being directed to the leaves, which is the place where important physiological processes and biochemical reactions occur.The high concentration of Na + in the absorption roots also prevented the tuberous roots from accumulating high concentrations of Na + .The yellowing and abscission of the older leaves suggest that there was a large accumulation of Na + and Cl -ions in this part of the plant, displaying an important strategy that protects the shoot apexes and the younger leaves.The high rate of leaf senescence in plants grown under 40 and 60 mM NaCl supports the hypothesis that there were higher Na + and Cl -concentrations in the older leaves.Munns, James, and Läuchli (2006) and Cruz et al. (2006) also confirmed that, in general, the concentrations of these two ions are much greater in older leaves than in younger leaves, which is called the exclusion mechanism; in certain circumstances, this concentration is so high that it can determine the senescence and death of older leaves.Thus, our data, when taken together, indicates that Na + was distributed mainly to the stem + petiole, absorption roots, and old leaves, which may have contributed to the reduction of the negative effects of salinity on cassava growth and dry matter accumulation.
It is worth noting that the amount of Na + in the leaves is an average, which includes the older leaves that are known to accumulate more Na + ; therefore, the presented amounts overestimate the concentrations of Na + in the younger leaves and underestimate the ones in older leaves.For some species, a Na + concentration of 10 mg kg -1 dry matter is not considered a toxic value (Silva, Ribeiro, Ferreira-Silva, Viégas, & Silveira, 2010).Thus, our data allows us to hypothesize that the Na + concentration found in the newly expanded cassava leaves was not at a toxic concentration.Hawker and Smith (1982) also observed that the leaf Na + concentration remained low in cassava leaf even with an application of 75 mM NaCl.

Growth and dry matter accumulation
The average size of each leaf, an indirect measure of leaf expansion, and the total number of leaves produced during the experimental period were not affected by salinity, even at 60 mM NaCl.This aspect may be related to the low concentration of Na + observed in this part of the plant.The highest NaCl concentration reduced leaf area, and this result was due solely to leaf abscission, which was one of the most affected parameters.This means that under salinity, the leaf production rate of cassava was lower than the abscission rate, which determined the smaller carbon assimilation areas; this is a negative aspect for plants growing under salt stress.The effects of salinity on the increase of abscission has also been described for other crops, such as strawberry (Orsini, Alnayef, Bona, Maggio, & Gianquinto, 2012) and tomato (Maggio, Raimond, Martino, & De Pascale, 2007).The drastic effects of salinity on leaf abscission have been explained by the increases in abscisic acid and ethylene concentrations that usually occur in organs that store high concentrations of Na + and/or Cl - (Arango, Wüst, Beyer, & Welsch, 2010;Amjad et al., 2014;Siddikee, Chauhan, & Sa, 2012).Salinity did not affect the differentiation process that leads to the formation of tuberous roots, demonstrating that this process, as well as the total number of leaves produced, was less sensitive to NaCl stress.
The total dry matter was linearly reduced with the increase of NaCl concentration in the growth medium.This decrease was primarily due to the reduction in tuberous roots dry matter since the absorbing roots dry matter was not reduced with salt stress, and the stem + petioles and leaves dry matter were reduced only at the highest NaCl concentration.That is, except for the tuberous roots, the other organs proved to be less sensitive to salt stress, even after accumulating large amounts of Na + in its tissues, such as that seen in the stem + petiole and absorption roots.This aspect indicates that cassava has the ability to isolate the ions Na + and Cl - from compartments associated with important metabolic processes.
The absorbing roots were less sensitive to salinity than the shoot components because NaCl did not reduce dry matter accumulation, even at 60 mM NaCl.Other authors have also shown that the shoot is more sensitive to salinity than the roots (Astolfi & Zuchi, 2013;Maggio et al., 2007;Carretero et al., 2007).The root:shoot ratio was affected only by the highest NaCl concentration in the growth medium.Thus, the reduction in the total dry matter of plants cultivated under the highest NaCl concentration may have been a consequence of the higher carbon allocation to the absorption roots than to the photosynthetic organs.The absorption roots growth maintenance under saline conditions is beneficial because they can accumulate large amounts of Na + , thus preventing a portion of this element from being transported to the leaves (Abideen et al., 2014), which was in accordance to what occurred in the present study.
Under the highest NaCl concentration, the tuberous roots accumulated the lowest concentrations of Na + at 2.56 g kg -1 dry matter, a value 84% lower than that in the stem + petiole; however, it was the organs that had the highest growth reduction.Even the 20 mM NaCl concentration reduced dry matter accumulation of the tuberous roots.In addition to the lower accumulation of Na + , cassava also not accumulate high concentrations of Cl -in their tuberous roots, as reported by Hawker and Smith (1982), who cultivated cassava under 50 mM NaCl.It can be inferred that root tuberization process is sensitive to the presence of those ions, even in low concentrations.
The lower photosynthetic rates of plants grown under saline conditions may have been one reason for the strong growth inhibition of tuberous roots.However, other factors may also be associated with this problem.According to Indira (1978), when cassava is grown under saline conditions, a degeneration of the cortical parenchyma and the formation of tyloses in the vessels of the tuberous roots occurs, inhibiting dry matter accumulation in these parts of the plant; according to the author, salinity delays the onset of tuberous roots formation, which may have caused less time for dry matter accumulation in these roots.Another problem is associated with the lower harvest index (HI), that provides a good indication of the balance between total carbon fixation and its distribution to the tuberous roots.The lower HI and the drastic reduction in dry matter accumulation in this organ seem to indicate that the flow and/or the use of carbon by tuberous roots were reduced by salt stress.In fact, the negative effects of salinity on the sink organs have been partly explained by damage to the transport and metabolism of sucrose (Balibrea, Cuartero, Bolarín, & Pérez-Alfocea, 2003) and starch formation and breakdown (Gimmler & Möller, 1981).Thus, the adverse effects of NaCl on the anatomy, the use of carbon, and the shorter time for filling, in addition to lower photosynthesis, may have been crucial to the lower growth of the tuberous roots.
Based on the shoot dry matter accumulation, cassava could be considered moderately tolerant to salinity.However, the drastic effects of NaCl on the tuberous roots dry matter indicates that, at this growth stage and for these experimental conditions, cassava behaves as a sensitive plant to salinity.Other studies have also classified cassava as moderately sensitive to salinity (Indira, 1978;Hawker & Smith, 1982).

Gas exchanges
Stomatal closure, as observed in this study, is a typical plant response to salt stress (Chaves, Flexas, & Pinheiro, 2009).The stomatal closure of salinized plants is attributed to lower leaf water potential and a reduction in relative water content, which results in a loss of cell turgor (Cabot, Sibole, Barceló, & Poschenrieder, 2014).However, the present study demonstrated that the leaf expansion rate, which may be indicative of turgor potential, was not affected by salinity, and the leaves of plants grown under 60 mM showed low Na + concentrations.Moreover, even when grown in severe water deficit conditions, cassava closed the stomata, but it did not significantly change its leaf water potential (El-Sharkawy, 2007).In addition, according to Alves and Setter (2004), signals derived from the roots and/or leaves could better explain stomatal movement of cassava grown under drought conditions than leaf water potential.All of these results seem to indicate that water relations are not the main determinants of stomatal closure in salinized cassava plants.The maintenance of good water status in cassava plants grown under moderate to high salinity has been observed by Carretero et al. (2007).
The highest NaCl concentration reduced E and this is a positive characteristic of plants grown under salinity because there is a positive relation between salt accumulation in the different organs and transpiration rate (Parihar, Singh, Singh, Singh, & Prasad, 2015).Thus, lower transpiration rates contributed to the lower accumulation of Na + , and possibly of Cl -, in the plant as a whole.The lower iWUE that occurred in plants grown under 40 mM NaCl was only due to a lower A, while for plants grown in 60 mM NaCl the lower iWUE was due to lower A and lower E. Lower iWUE have been associated with lower plant tolerance to salinity (Omamt, Hammes, & Robbertse, 2006).
The carbon assimilation rate (A) was reduced even under 40 mM NaCl, which was considered an intermediate level of stress.This result was more drastic in the older leaves than in the newly expanded leaves.It is worth noting that this concentration of NaCl did not reduce the growth of the shoots and absorption roots or reduce leaf expansion, showing that, except for tuberous roots, the photosynthetic process was more sensitive to NaCl concentration than the growth parameters.The lower stomatal conductance of the plants grown under salinity was essential to a lower A, as can be deduced from the correlation between g s and A that was high and positive.This result imply that the restriction of CO 2 diffusion from the outside air into the chloroplast was one of the factors responsible for lower A, as was the case of the newly expanded leaves of the plants grown with 40 mM NaCl, which showed lower intercellularCO 2 concentrations.All other saline treatments showed an increase in intercellular CO 2 concentration, clearly indicating that in addition to lower stomatal conductance, salinity may have increased mesophilic resistance to the ingress of atmospheric CO 2 into carboxylation sites and/or decreased the enzymatic activities associated with photosynthetic carbon metabolism, a common effect in plants grown under saline conditions (Chaves et al., 2009;Pérez-López et al., 2012).Another important aspect, as deduced from a lower HI, is that the tuberous roots' ability to metabolize sucrose unloaded from phloem could have been impaired, leading sugar accumulation in the source leaves, which can determine photosynthesis inhibition, a process known as "photosynthetic repression feedback".This has already been observed in cassava by Cruz, Mosquim, Pelacani, Araujo, and DaMatta (2003).Albacete et al. (2014) also indicated that the reduction in sink activity might be an indirect effect of salinity on A. Thus, the lower photosynthesis in the leaves of cassava plants grown under salinity was due to stomatal and, most likely, to biochemical factors.The probable accumulation of sugars in the leaves may have also been one of the components that led to the stomatal closure of plants grown under salinity.In fact, the importance of sucrose and other compounds derived from cellular metabolism in the regulation of stomatal movement has been well-established (Lawson, Simkin, Kelly, & Granot, 2014).These assumptions, however, need to be further investigated with salinized cassava plants.
It is most likely that the drastic reduction of photosynthesis in older leaves, compared to the newly expanded leaves, is related to Na + and Cl -ion accumulation at toxic levels, a situation that normally occurs when plants are grown under high salt conditions (Cruz et al., 2006;Munns & Tester, 2008).Indeed, it has been shown that A is inversely proportional to the concentration of Na + and Cl -in leaf tissue (Wang et al., 2015).These older leaves were also more yellow, indicating photosynthetic pigment loss and probably protein reduction (Qiao et al., 2010), which likely contributed to the reduction of A in these leaves.

Conclusion
Our results indicate that salt stress affects carbon assimilation in cassava plants via its effects on stomatal resistance and its negative interference on the biochemical reactions that occur in chloroplast stroma.The detrimental effect of salt stress on the dry matter of cassava plants is associated with an alteration of the sink-source relationships, lower carbon assimilation, and the high sensitivity of tuberous roots to NaCl.In this respect, the tuberous roots could be the target organ in studies that aim to improve the tolerance of cassava to salinity.

Figure
Figure 4. Harv concentrations and means fo different at p <